KR102113778B1 - Myocardial-like structures prepared using porous supports method for evaluating drug toxicity using the same - Google Patents

Abstract

The present invention relates to a myocardial-like structure, and more particularly, to a myocardial-like structure prepared by simultaneously culturing a porous support and stem cell-derived myocardial cells and a method for evaluating drug toxicity using the same. The myocardial-like structure produced by using the porous natural polymer support according to the present invention not only pulsates but also has the function of the myocardium, and there is a change in contractility when treating the myocardial-like structure with a drug-free buffer. In this case, it was confirmed that the contractile force is restored, and thus it can be used in various fields in new drug development, disease research, and artificial organ development.

Description

Myocardial-like structures prepared using porous supports method for evaluating drug toxicity using the same}

The present invention relates to a myocardial-like structure, and more particularly, to a myocardial-like structure prepared by simultaneously culturing a porous support and stem cell-derived myocardial cells and a method for evaluating drug toxicity using the same.

3D organ analogs are cultured from materials such as Matrigel that mimic extracellular matrix, and this 3D culture technique has very few nuclear changes and does not have a spontaneous cancer process, unlike the conventional 2D cell culture method. Is being reported. Therefore, long-term analog culture is considered to be advantageous for long-term preservation of characteristics in the body over any cell culture method presented to date. Recently, the 3D organ simulation organ analogue is expected to be useful in the field of precision medicine in the future because it can be an essential disease simulation model in implementing patient-specific treatment as well as basic research of disease understanding. More specifically, it will be possible to understand the characteristics of diseases with different constellations for each patient, discover the drugs that can be most efficient using a long-term analog platform, and apply them to customized treatment.

Accordingly, the present inventors completed the present invention by preparing a myocardial-like structure prepared by co-culturing a porous support and stem cell-derived cardiomyocytes, and developing a drug toxicity evaluation method using the same.

Accordingly, an object of the present invention is to provide a myocardial-like structure prepared using a porous natural polymer support, a method for manufacturing the same, and a method for testing activity or toxicity of a test substance using the same.

In order to achieve the above object, the present invention provides a method for preparing a myocardial structure comprising the step of simultaneously culturing a porous natural polymer support and cardiomyocytes.

In addition, the present invention provides a myocardial-like structure produced by the above manufacturing method.

In addition, the present invention provides a method for testing the activity or toxicity of a test substance, comprising the step of treating a test substance with a myocardial-like structure and measuring the contractility of the myocardial-like structure.

The myocardial-like structure produced by using the porous natural polymer support according to the present invention not only pulsates but also has the function of the myocardium, and there is a change in contractility when treating the myocardial-like structure with a drug-free buffer. In this case, it was confirmed that the contractile force is restored, and thus it can be used in various fields in new drug development, disease research, and artificial organ development.

1 is a view showing a method of manufacturing a myocardial structure according to the present invention.
FIG. 2 is a view showing the results of observing a myocardial structure using a scanning electron microscope (SEM) image.
3 is a diagram showing the results of confirming the cell viability according to the period of the myocardial cells attached to the myocardial-like structure as a survival / killing analysis method.
4 is a view showing the results of observing the organelles of the myocardial cells attached to the myocardial structure using a transmission electron microscope (TEM) image.
5 is a view showing the results of checking the heart rate and heart rate of the myocardial structure according to the present invention.
6 is a diagram showing the results of evaluating cardiac toxicity of nifedipine, a calcium channel inhibitor, using the myocardial structure according to the present invention.
7 is a view showing the results of evaluating the cardiac toxicity of the calcium channel inhibitor isoprenaline using the myocardial structure according to the present invention.
8 is a view showing the results of evaluating the cardiac toxicity of the cardiotoxic drug terpenadin using the myocardial structure according to the present invention.
9 is a view showing the results confirming the resilience of the myocardial-like structure according to the present invention reacted with the drug.
10 is a view showing the results of analyzing the morphology of the myocardial-like structure according to the present invention by the hematoxylin & eosin staining method.
11 is a view showing the results of confirming the maturity of the myocardial-like structure according to the present invention by a tissue immunochemical staining method.

Hereinafter, the present invention will be described in detail.

According to an aspect of the present invention, the present invention provides a method for producing a myocardial-like structure comprising the step of simultaneously culturing a porous natural polymer support and cardiomyocytes.

In the present invention, "natural polymer" means a polymer material existing in nature or produced by an organism. Examples of the natural polymer include, but are not limited to, collagen, fibronectin, gelatin, chitosan, alginic acid and hyaluronic acid.

In a preferred embodiment of the present invention, cardiomyocytes were cultured using porous collagen.

In the present invention, "myocardium" is a special type of muscle constituting the middle layer of the heart wall of a vertebrate animal, the structure of which is a rhabdomyo muscle or a mosaic-like muscle belonging to a smooth muscle.

In one embodiment of the present invention, the cardiomyocytes are preferably stem cell-derived cardiomyocytes, and more preferably cardiomyocytes derived from induced pluripotent stem (iPS), but are not limited thereto.

In the present invention, "myocardial-like structure" is a long-term analogue produced by culturing or recombining three-dimensional myocardial cells derived from stem cells, and can be used for new drug development, disease treatment, and artificial organ development. When developing a new drug, the existing drug test had a limitation in that the results of animal and clinical experiments were different, such as side effects not found in animal experiments were found in humans. The myocardial structure is expected to overcome these limitations, and it can also escape the ethical controversy over animal testing. In addition, since it is possible to produce similar organs by culturing the patient's cells, it is attracting attention as a methodology for personalized clinical trials, and may be used for personalized organ development in the future.

In one embodiment of the present invention, the culture is preferably cultured for 32 to 38 days, and more preferably for 35 days. When the myocardial cells are cultured for less than 32 days, the rhythm interval is irregular and the rhythm is slow, and when cultivated for more than 38 days, the rhythm and the rhythm interval are irregular, which is not suitable for use as a myocardial structure.

In another aspect of the present invention, a myocardial-like structure produced by the above method is provided.

The myocardial-like structure according to the present invention is that iPS-derived cardiomyocytes are attached to a porous collagen support, and multinucleated cells and a robust junction are observed on the 32 to 38 days of incubation. The myocardial-like structure has a change in contractile force when the test substance is processed, and when the drug-free buffer is treated, the contractile force is restored, and the effect of repeated or sequential treatment of the test substance or other test substance can be evaluated. Therefore, the myocardial structure can be used in new drug development, disease research, and artificial organ development.

According to another aspect of the present invention, the present invention provides a method for testing the activity or toxicity of a test substance comprising treating a test substance in a myocardial-like structure and measuring contractility of the myocardial-like structure.

In the present invention, the "method for testing activity or toxicity" does not limit its type, and is preferably a contractile-based cardiotoxicity evaluation method when testing for activity or toxicity using a myocardial structure analog.

In one embodiment of the present invention, the contractile force measurement is preferable to measure the change in the length, area, or rhythm of the myocardial-like structure, but is not limited thereto.

In another embodiment of the present invention, the activity of the test substance is preferably a drug metabolic activity measurement or drug interaction evaluation, but is not limited thereto.

Hereinafter, the present invention will be described in more detail through examples. These examples are only for illustrating the present invention, it will be apparent to those skilled in the art that the scope of the present invention is not to be construed as limited by these examples.

Example  1. Fabrication of a myocardial-like structure using a porous collagen support

A three-dimensional culture system was constructed using a porous collagen support, and the three-dimensional culture system has a better structure than cultured in a commonly used two-dimensional environment, and thus interacts more closely with neighboring cells and forms a three-dimensional form. Keep it better. A method of fabricating a myocardial structure using a porous collagen support is briefly illustrated in FIG. 1.

First, for the preparation of a myocardial structure, human iPS-derived cardiomyocytes (iCell® Cardiomyocytes, CMC-100-010-001, USA, Cellular Dynamics International) were prepared. In the experiment including the fabrication of a myocardial structure, cardiomyocytes were cultured for 7 days or more by attaching cells to a culture plate coated with gelatin or collagen. The culture was performed using a plating medium (Plating Medium: Plating Medium 50%, FBS (Hyclone, SH30919.03, USA) 10%), and was performed in a 37 ° C incubator containing 5% carbon dioxide and saturated water vapor. Medium was changed 2-3 times a week.

A porous collagen support (CollaCote Dressing, Zimmer Biomet) having a size of 2x4 cm was cut into a 5x5x2mm size using a razor. The cultured iPS-derived cardiomyocytes were treated with 0.25% trypsin-EDTA to float as single cells, collected in 50 mL centrifuge tubes, and centrifuged at 1000 rpm for 3 minutes. The supernatant medium of the tube was removed by aspiration, and the cell pellet was resuspended by adding fresh medium. Resuspended cells were counted. 20 μl of the cell suspension was dispensed onto a porous collagen support and transferred to a clean Petri dish. The porous collagen support in which the cell suspension was dispensed was cultured at 37 ° C. in a 5 to 7% CO 2 incubator for 3 hours. In order to prevent the cells from drying out, maintenance medium (Maintain Medium: Maintain medium 90%, FBS 10%) was dispensed on the collagen support by 10 µl every 30 minutes during the culture. The myocardial-like structure was completed by filling the maintenance medium so that the porous collagen support to which the cells were attached was sufficiently submerged in the medium. The myocardial structure was confirmed using an optical microscope, and the maintenance medium was replaced every 2-3 days.

Example  2. Scanning electron microscope ( SEM ) Myocardial structure observation using images

Using a scanning electron microscope (SEM), the surface of the iPS-derived cardiomyocytes to ^{0.5x10 7 / ㎖, 1x10 7 /} ㎖, 2x10 7 / ㎖ concentration of the culture solution 20㎕ the injection to the production of a structure and diluted with similar myocardial The shape was observed.

Specifically, the myocardial structure was fixed in a low temperature environment of 4 ° C using 2.5% glutaraldehyde (in PBS) at 1, 3, and 5 weeks. The immobilized myocardial structure was washed with 0.1M phosphate buffer solution (pH7.4) for 10-20 minutes. After post-fixing, the mixture was advanced with 1% OsO ₄ (osmic acid) for about 1 hour, and washed with 0.1M phosphate buffer solution (pH7.4) again. Subsequently, the sample was dehydrated in tert-butyl alcohol and then substituted using ethanol. After drying to remove the dehydration solution contained in the sample after dehydration, the sample was mounted on an aluminum stub with an adhesive tab and coated with gold having a thickness of ˜30 nm to observe the sample. As a control, a collagen support without cells was used. The results of observing the myocardial structure using a scanning electron microscope are shown in FIG. 2.

As shown in FIG. 2, it was observed that pores of various sizes were formed between irregular collagen skeletons and iPS-derived cardiomyocytes were attached to the collagen support. In addition, it was observed that as the number of cells was injected, the myocardial cells were arranged in a line in the collagen support at an early stage. As time passed, it was confirmed that the cell-cell bond was firm and that several cardiomyocytes were joined together rather than a single cell.

Example  3. Attached to myocardial-like structures through survival / kill analysis Myocardial  Check cell viability by period

The cell viability of iPS-derived cardiomyocytes attached to a myocardial-like structure was confirmed. Specifically, iPS-derived cardiomyocytes were cultured for 42 days by injecting 20 μl of a culture solution diluted to a concentration of 0.5 × 10 ⁷ / mL, 1 × 10 ⁷ / mL, and 2 × 10 ⁷ / mL. During the culture, a live-dead assay (abcam, ab65470) was performed at weekly intervals from the 4th day of culture. Survival / kill analysis was performed according to the manufacturer's manual, and the results are shown in FIG. 3.

3, it was confirmed that most of the cells survived regardless of the number of inoculated cells or the culture period. In FIG. 3, green fluorescence means living cells, and red fluorescence means dead cells.

Example  4. Transmission electron microscope ( TEM ) Attached to myocardial-like structures using images Myocardial Organelle  observe

To confirm the degree of maturation in the organelles of the myocardial cells attached to the myocardial structure, iPS-derived myocardial cells were cultured with 20 µl of diluted culture solution at a concentration of 0.5x10 ⁷ / ml, 1x10 ⁷ / ml, 2x10 ⁷ / ml. The myocardial structure prepared by injection was observed with a transmission electron microscope. Specifically, 2.5% glutaraldehyde (in PBS) was used to fix in a low temperature environment of 4 ° C. The fixed collagen sponge was washed with 0.1M phosphate buffer (pH7.4) for 10-20 minutes. After post-fixing, the mixture was advanced with 1% OsO ₄ (osmic acid) for about 1 hour, and washed with 0.1M phosphate buffer (pH7.4) again. In order to remove moisture in the sample, dehydration of 50%, 70%, 80%, and 95% 100% ethyl alcohol was performed at a low concentration to a high concentration within 5 minutes. After cutting to 1 µm using an ultra-micro-slicing machine (Ultra Microtome), it is transferred to a slide glass and attached and fixed while being stretched in a hot plate (80 ° C). After the electron staining, it was observed through a transmission electron microscope, and the results are shown in FIG. 4.

As shown in FIG. 4, some myofibrillar fibers were observed at week 1, and mature mitochondria and myofibrillar fibers were observed at week 3 as compared to week 3 and at week 5. In addition, it was confirmed that solid junctions were formed with the polynuclear cells found in mature cardiomyocytes.

Example  5. Myocardial structure Heart rate  And beat rate check

iPS-derived cardiomyocytes were injected with 20 μl of a culture solution diluted to a concentration of 0.5 × 10 ⁷ / mL, 1 × 10 ⁷ / mL, and 2 × 10 ⁷ / mL to confirm the heart rate and beat interval of the myocardial-like structure prepared. Specifically, the heart rate and beat interval per minute of the artificial myocardial structure from the 4th day of culture to the 42nd day at weekly intervals were measured through video shooting. The results of checking the heart rate and the heart rate are shown in FIG. 5.

As shown in Figure 5, the myocardial structure was confirmed that the pulsation interval is not constant between 2 and 5 seconds at the beginning of culture and the pulsation is slow. In the myocardial structure, it was confirmed that the heart rate increased the most at 35 days, and the beat interval became constant. The myocardial structure after 35 days of culture was confirmed to have irregular heart rate and beat interval. The above results indicate that the 5th week post-cultivation myocardial-like construct is best used as an in vitro model for evaluating cardiac toxicity.

Example  6. Evaluation method of cardiac toxicity based on contractility using myocardial structure

Using a myocardial-like structure, a systolic-based cardiac toxicity assessment was performed.

The effects of drugs on cardiac contractility by measuring the following parameters related to contractility using the video-based IonOptix's edge detector system were evaluated indirectly through the size, speed, area, etc. of the length change of the myocardial structure. . Using cardiac toxic drugs terpenadin and nifedipine with a negative inotropic effect and isoprenaline with a positive inotropic effect as a calcium channel inhibitor, the contractility of myocardial-like structures Drug reactivity was confirmed. The specific method for measuring shrinkage is as follows (i) to (vi).

(i) After incubating the myocardial-like structure for about 35 days, placing it in a recording chamber for measuring contractile force, and stabilizing the buffer solution at 37 degrees by perfusion for 10 minutes or more at a rate of about 3 mL / min.

Reagent mol conc. (mM) NaCl 141.4 KCl 4 NaH ₂ PO ₄ 0.33 HEPES 10 MgCl ₂ One CaCl ₂ 1.8 Glucose 5.5 Mannitol 14.5

(ii) Using a stimulator to stimulate for 10 minutes or more at 10 V, 1 Hz to stabilize myocardial-like structures.

(iii) Manipulate the handle of the MyoCam-S video camera to find a suitable measurement site for the myocardial structure and focus the measurement site on the video display screen of the IonWizard software.

(iv) When stimulation was performed at 1, 2, and 3 Hz at 10 V using a stimulator and the myocardial structure was measured, it was confirmed that it was adjusted according to the size of the stimulus. This can be judged to have a function similar to that of a real heart.

(v) Perfusion of the buffer solution, record the control reaction under the untreated condition, and flow a 10 nM isoprenaline solution, a sympathetic stimulant, for 3 to 10 minutes until the reaction is constant to measure the response of the drug. While measuring the shrinkage of the support. After drug treatment, signals of arrhythmia are detected.

(vi) Drugs of various concentrations are sequentially processed from low to high concentrations until the drug response is constant for 3-10 minutes and the parameters related to the contractile force in the drug reaction conditions are recorded to control the drug against the collagen support. Recorded and analyzed the effect on contractility.

The results of evaluating cardiac toxicity based on contractility using a myocardial structure are shown in FIGS. 6 to 8.

6 to 8, the myocardial-like structures treated with 0.1 and 1 μM nifedipine showed negative denaturation in a concentration-dependent manner, and the myocardial-like structures treated with 10 and 100 nM isoprenaline were positively dependent in a concentration-dependent manner. It was confirmed to show. In addition, it was confirmed that the myocardial structure treated with terpenadin at concentrations of 10, 100, 300, and 1000 nM showed positive denaturation in a concentration-dependent manner after the control record.

Next, the resilience of the myocardial-like structure reacted with the drug was confirmed. Specifically, nifedipine was sequentially treated from low concentration to high concentration until the drug reaction became constant for 3-10 minutes, and the contractile force was observed under drug reaction conditions. Then, the mixture was stabilized by perfusion for 20 minutes at a rate of 3 mL / min with a buffer containing no drug. The results of confirming the resilience of the myocardial-like structure reacted with the drug are shown in FIG. 9.

As shown in Figure 9, as a result of perfusion of the drug-free buffer in the myocardial-like structure reacted with the drug, it was confirmed that the contractile force of the myocardial-like structure is restored. This is a phenomenon caused by pharmacological activity due to the pharmacokinetics of absorption, distribution, metabolism, and excretion when drugs enter the body. The above results indicate that it is possible to measure effects such as excretion even in a myocardial-like structure produced in vitro, and thus predict a more accurate cardiotoxic response of the drug.

Example  7. Verification of myocardial-like structures using tissue immunochemical staining

iPS-derived cardiomyocytes injected into 20 μl of a culture solution diluted to a concentration of 0.5x10 ⁷ / ml, 1x10 ⁷ / ml, and 2x10 ⁷ / ml into a porous collagen support cut into 5x5x2mm size, and a conventionally known method of 3D artificial myocardial structure Hematoxylin & Eosin (H & E) staining was performed to perform morphological analysis, and the results are shown in FIG. 10.

As shown in Fig. 10, it was observed that the myocardial cells were arranged in a line on the collagen support. As time passed, it was confirmed that the cell-cell bond was firm and that several cardiomyocytes were joined together rather than a single cell.

To confirm the difference in myocardial cell maturity and protein expression over time, tissue immunochemical staining was performed over 1, 3, and 5 weeks. Specifically, a mold was manufactured using OCT and frozen at -80 ° C. Using a frozen tissue slicer, a cross section of the porous collagen support cut from the frozen mold was obtained. The frozen tissue slicer is an equipment capable of producing tissue slices for histological examination immediately after freezing without fixation by a chemical fixation solution, and is an important equipment for performing histochemical and histological immunological studies. Myocardial-like structure fragments were transferred onto a glass slide and observed with a confocal fluorescence microscope. Whether the expression of a gap junction protein such as connectin-43 was expressed from the myocardial-like structure fragment and the myocardial cell maturity was confirmed using a myocardial cell specific marker such as actin. The results of observing the myocardial structure are shown in FIG. 11.

As shown in FIG. 11, α-actin was identified as green fluorescence, connectin-43 as red fluorescence, and nuclei as DAPI staining as blue fluorescence. From week 1 to week 5, myocardial cells present in collagen support showed increased expression of α-actin and connectin-43. Among them, α-actin showed a more mature nodular form at weeks 3 and 5 when compared to week 1. It can be seen that the above results indicate that the heart rate and beat interval of the myocardial structure, which are the results of Example 5, are constant.

Overall, the present inventors verified the beat and function of a myocardial-like structure produced using a porous collagen support. In addition, it was confirmed that when the drug is treated with the myocardial structure, the contractile force is changed, and when the buffer containing no drug is treated, the contractile force is restored. Therefore, the myocardial-like structure of the present invention can be used in various fields in the field of new drug development, disease research, and artificial organ development.

As described above, since a specific part of the present invention has been described in detail, it is obvious to those skilled in the art that this specific technique is only a preferred embodiment, and the scope of the present invention is not limited thereby. something to do. Therefore, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

Claims (9)

A method of preparing a myocardial-like structure for testing the activity or toxicity of a test substance, including; simultaneously culturing a porous collagen support and cardiomyocytes for 32 to 38 days.
delete According to claim 1,
The myocardial cell is derived from stem cells, manufacturing method.
According to claim 3,
The stem cells are induced pluripotent stem cells (induced pluripotent stem, iPS), the production method.
delete A myocardial-like structure produced by the method according to any one of claims 1, 3 and 4.
Treating a test substance in a myocardial-like structure according to claim 6; And
Measuring the contractile force of the myocardial-like structure; including, a method of testing the activity or toxicity of the test substance.
The method of claim 7,
The measurement of the contractile force is to measure the change in the length, area, or rhythm of the myocardial structure, and test the activity or toxicity of the test substance.
The method of claim 7,
The activity is a method for testing the activity or toxicity of a test substance, which is a measurement of drug metabolism activity or evaluation of drug interaction.

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